Virtually all aspects of RNA metabolism involve DExH/D proteins, a large and highly conserved class of enzymes. Numerous proteins from this family play direct roles in disease states including tumorogenesis and infectious diseases. DExH/D proteins comprise the DEAD-box, the DExH and the DEAH subgroups and enzymes from all three subgroups couple ATP binding and/or hydrolysis to RNA unwinding or structural changes in RNA-protein complexes. Despite the conservation of structure and sequence within the DExH/D proteins, it has recently become clear that fundamental functional differences exist between the DExH/D subgroups. DEAD-box proteins, the largest DExH/D protein subgroup, have been found to unwind duplexes not by translocation, like previously studied helicases, but by ATP-driven, local strand separation. Mechanistic concepts were developed for translocating helicases thus do not apply to DEAD-box proteins. Here, it is proposed to define DEAD-box protein function on the molecular level. First, a kinetic and thermodynamic framework for RNA unwinding by the DEAD-box protein Ded1p will be developed, with the aim to understand how this protein couples ATP binding and hydrolysis to conformational work on RNA. This framework will then be utilized to analyze two additional, different DEAD-box proteins, Mss116p, and eIF4A. A quantitative, comparative analysis between all three enzymes is performed, to gain insight into the functional diversity of DEAD-box proteins. Finally, as the first step towards understanding the function of DEAD-box proteins in more complex physiological environments, it is investigated how physiological co-factors modulate the mechanism(s) of Ded1p and eIF4A. The proposed study combines biochemical and biophysical ensemble methods with single molecule techniques. The work will not only provide unique insight into the molecular mechanism of DEAD- box proteins but also conceptually and methodologically advance quantitative analysis and understanding of RNA-protein interactions. DExH/D proteins are a large class of enzymes essential for gene expression, but their function is not well understood. Many of these enzymes have been implicated in disease states including cancer and infectious diseases. To provide critical insight into the molecular basis of these diseases and to guide the development of potential therapeutic agents, we propose to study the mechanism of DExH/D proteins.